Catalytic anti-factor VIII allo-antibodies

ABSTRACT

The present invention relates to a method of determining the presence of catalytic anti-Factor VIII allo-antibodies capable of degrading Factor VIII in a mammal, and of characterising the cleavage sites in said Factor VIII molecule by said catalytic anti-Factor VIII allo-antibodies. It also relates to an anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor; and to a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination; and further to a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor. Finally, the present invention relates to the application in therapeutics of said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor, of a pharmaceutical composition comprising said catalytic anti-Factor VIII allo-antibodies which are capable of degrading Factor VIII and which originate from said method of determination, and of a pharmaceutical composition comprising said anti-Factor VIII allo-antibody-catalysed Factor VIII degradation inhibitor.

FIELD OF THE INVENTION

The present invention relates to a method of determining the presence ofcatalytic anti-Factor VIII allo-antibodies capable of degrading FactorVIII in a mammal, and of characterising the cleavage sites in saidFactor VIII molecule by said catalytic anti-Factor VIII allo-antibodies.

The present invention also relates to an anti-Factor VIIIallo-antibody-catalysed Factor VIII degradation inhibitor.

The present invention further relates to a pharmaceutical compositioncomprising said catalytic anti-Factor VIII allo-antibodies which arecapable of degrading Factor VIII and which originate from said method ofdetermination, and to a pharmaceutical composition comprising saidanti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor.

Finally, the present invention relates to the application intherapeutics of said anti-Factor VIII allo-antibody-catalysed FactorVIII degradation inhibitor, of a pharmaceutical composition comprisingsaid catalytic anti-Factor VIII allo-antibodies which are capable ofdegrading Factor VIII and which originate from said method ofdetermination, and of a pharmaceutical composition comprising saidanti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor.

BACKGROUND TO THE INVENTION

Haemophilia A is an X chromosome-linked recessive disorder resulting indefective or deficient Factor VIII molecules, which, in its severe form,is a life-threatening and crippling haemorrhagic disease.

Infusion of homologous Factor VIII to patients with severe haemophilia Aresults, in 25% of the cases, in the emergence of anti-Factor VIIIallo-antibodies (Ehrenforth, S., Kreuz, W., Scharrer, I., Linde, R.,Funk, M., Güngör, T., Krackhardt, B. and Kornhuber, B., <<Incidence ofdevelopment of factor VIII and factor IX inhibitors in haemophiliacs>>,Lancet, 1992, 339: 594-598), that inhibit Factor VIII procoagulantactivity by steric hindrance of the interaction of Factor VIII eitherwith stabilising molecules (Saenko, E. L., Shima, M., Rajalakshmi, K. J.and Scandella, D., <<A role for the C2 domain of factor VIII in bindingto von Willebrand factor>>, J. Biol. Chem., 1994, 269: 11601-11605; andSaenko, E. L., Shima, M., Gilbert, G. E., and Scandella, D., <<Slowedrelease of thrombin-cleaved factor VIII from von Willebrand factor by amonoclonal and a human antibody is a novel mechanism for factor VIIIinhibition>>, J. Biol. Chem., 1996, 271: 27424-27431), with moleculesessential for its activity (Arai, M., Scandella, D., and Hoyer, L. W.,<<Molecular basis of factor VIII inhibition by human antibodies:Antibodies that bind to the factor VIII light chain prevent theinteraction of factor VIII with phospholipid>>, J. Clin. Invest., 1989,83: 1978-1984; and Zhong, D., Saenko, E. L., Shima, M., Felch, M. andScandella, D., <<Some human inhibitor antibodies interfere with factorVIII binding to Factor IX>>, Blood, 1998, 92: 136-142), or withactivating molecules (Lubahn, B. C., Ware, J., Stafford, D. W., andReiser, H. M., <<Identification of a FVIII epitope recognized by a humanhemophilic inhibitor>>, Blood, 1989, 73: 497-499; and Neuenschwander, P.F., and Jesty, J., <<Thrombin-activated and factor Xa-activated humanfactor VIII: differences in cofactor activity and decay rate>>, Arc.Biochem. Biophys., 1992, 296: 426-434).

SUMMARY OF THE INVENTION

In an entirely surprising way, a discovery has been made by theApplicants of a degradation of Factor VIII by allo-antibodies of twohigh responder patients with severe haemophilia A, demonstrating aheretofore unknown mechanism by which Factor VIII inhibitors may preventthe pro-coagulant function of Factor VIII.

The Applicant's discovery of catalytic anti-Factor VIII allo-antibodiesis to the best of his knowledge the first report on the emergence ofcatalytic antibodies that are INDUCED upon treatment of patients withFactor VIII. It was heretofore considered very surprising, even absurdor unbelievable, that antibodies are formed, in the presence of FactorVIII, which would actually render the Factor VIII molecule inactivethrough catalytic hydrolysis (

proteolysis

). However, the catalytic antibodies reported so far, are allauto-antibodies found in the course of a disease process or inphysiological conditions. Thus, induced antibodies are calledALLO-antibodies, the origin of which is clearly different from theorigin of AUTO-antibodies in any auto-immune disease.

The calculated average Km and apparent Vmax for the reaction ofanti-Factor VIII antibodies of one of the patients were 9.46±5.62 μM and85±60 fmol.min⁻¹, respectively. The kinetic parameters of Factor VIIIhydrolysis suggest a functional role for the catalytic immune responsein the inactivation of Factor VIII in vivo.

The characterisation of anti-Factor VIII allo-antibodies assite-specific proteases hence provide new approaches to the treatment ofdiseases of a patient who possess anti-Factor VIII allo-antibodies.

Thus, according to a first aspect, the present invention provides amethod of determining the presence of catalytic anti-Factor VIIIallo-antibodies capable of degrading Factor VIII in a mammal,characterised in that it comprises:

-   -   i) isolating the plasma from a sample of blood taken from said        mammal,    -   ii) isolating anti-Factor VIII allo-antibodies from said plasma;    -   iii) placing said anti-Factor VIII allo-antibodies in contact        with Factor VIII for a period of time sufficient to permit any        degradation of said Factor VIII by said anti-Factor VIII        allo-antibodies; and    -   iv) determining, after said period of time, whether said Factor        VIII has effectively been degraded by said anti-Factor VIII        allo-antibodies.

According to an embodiment of step ii) of the method of the presentinvention, said anti-Factor VIII allo-antibodies are isolated from saidplasma by combining them with said Factor VIII, said Factor VIII beingpreferably coupled to a matrix. Advantageously, in step ii), saidanti-Factor VIII allo-antibodies are isolated by affinitychromatography. Preferably, in step ii), said affinity chromatographycomprises the use of a Sepharose matrix, preferably activated withcyanogen bromide.

According to an embodiment of step iii) of the method of the presentinvention, said Factor VIII is labelled with a labelling agent,preferably a radio-labelling agent, such as ¹²⁵I in particular.Advantageously, in step iii), said Factor VIII is placed in contact withthe anti-Factor VIII allo-antibodies for a period of time of betweenabout 0.5 and about 30 hours, preferably about 10 hours, at atemperature of about 15 to about 40° C., preferably 38° C.

According to an embodiment of step iv) of the method of the presentinvention, the determination of whether said Factor VIII has effectivelybeen degraded by said anti-Factor VIII allo-antibodies is carried out bya determination comprising a separation technique, such as gelelectrophoresis, such as SDS PAGE in particular, or gel filtration, suchas fast protein liquid chromatography gel filtration in particular, anda visualisation technique, such as autoradiography in particular.

In accordance with a further embodiment of the method of the presentinvention, said method is characterised in that it further comprises:

-   -   v) characterising the site(s) in said Factor VIII molecule        cleaved by said anti-Factor VIII allo-antibodies.

According to an embodiment of step v) of the method of the presentinvention, said characterisation is carried out by placing said FactorVIII in contact with said anti-Factor VIII allo-antibodies capable ofdegrading Factor VIII, separating and then sequencing the fragments ofFactor VIII resulting therefrom. Advantageously, said separation iscarried out using a technique such as gel electrophoresis, such as SDSPAGE in particular, or gel filtration. Said sequencing is advantageouslycarried out using a technique such as N-terminal sequencing, such as byusing an automatic protein microsequencer in particular. By using thesaid sequencing, the following scissile bonds are located:Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr¹⁶⁸⁰-Asp¹⁶⁸¹,located on the N-terminus of the A3 domain, and Glu¹⁷⁹⁴-Asp¹⁷⁹⁵ locatedwithin the A3 domain of the Factor VIII molecule.

According to a second aspect, therefore, the present invention providesan amino acid sequence:

Ser Val Ala Lys Lys His Pro;an amino acid sequence:

Asp Glu Asp Glu Asn Gln Ser; andan amino acid sequence:

Asp Gln Arg Gln Gly Ala Glu.

The present invention also extends to variants or analogues of this orany other sequence of Factor VIII which are capable of inhibiting anysite in the Factor VIII molecule which is susceptible to being lysed byan anti-Factor VIII allo-antibody. Within the context of the presentinvention, such a variant can be, for example, a peptide or non-peptideanalogue of an amino acid sequence described supra which inhibits anysite in the Factor VIII molecule which is susceptible to being lysed byan anti-Factor VIII allo-antibody. Such a variant can be, for example, avariant of the sequence which is either shorter by a few amino acids, atthe N-terminal, the C-terminal, or both termini, for example, or longerby a few amino acids (it being possible to obtain such variants bychemical synthesis or by enzymatic digestion of the naturally occurringmolecule), so long as the variant inhibits any site in the Factor VIIImolecule which is susceptible to being lysed by an anti-Factor VIIIallo-antibody.

Hence, according to a third aspect, the present invention provides ananti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor. Advantageously, this inhibitor is characterised in that itcomprises a protease inhibitor. Examples of protease inhibitors that canbe used as anti-Factor VIII allo-antibody-catalysed Factor VIIIdegradation inhibitors within the context of the present invention,without being limited thereto, are fluorophosphate-type inhibitors, suchas DFP for example, or sulphonyl fluoride-type inhibitors, such as PMSFor AEBSF (4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride(notably marketed by Roche Diagnostics GmbH, Mannheim, Germany, underthe trademark Pefabloc®)), for example. More particularly, thisinhibitor is characterised in that said inhibitor inhibits cleavage ofthe scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1 domains,Tyr¹⁶⁸⁰-Asp¹⁶⁸¹, located on the N-terminus of the A3 domain, andGlu¹⁷⁹⁴-Asp¹⁷⁹⁵ located within the A3 domain of the Factor VIIImolecule. More preferably still, this inhibitor is characterised in thatit comprises a peptide or non-peptide analogue of the amino acidsequence:

Ser Val Ala Lys Lys His Pro;a peptide or non-peptide analogue of the amino acid sequence:

Asp Glu Asp Glu Asn Gln Ser; ora peptide or non-peptide analogue of the amino acid sequence:

Asp Gln Arg Gln Gly Ala Glu.

The Factor VIII degradation inhibitors as defined supra, as well astheir addition salts, in particular their pharmaceutically acceptableaddition salts, have a very valuable pharmacological profile in thatthey possess neutralising activity towards anti-Factor VIIIallo-antibodies.

These properties justify their application in therapeutics and theinvention further relates, by way of drugs, to the Factor VIIIdegradation inhibitors above, as well as their addition salts, inparticular their pharmaceutically acceptable addition salts.

They will therefore be particularly indicated in the treatment ofdiseases of, inter alia, haemophilic nature, more particularly diseasesinvolving coagulation defects due to Factor VIII insufficiency.

An example of their use which may be mentioned is the treatment of highresponder patients with diseases such as mild or severe haemophilia A,for example (in the case in which catalytic antibodies are found inthese patients), on the one hand, and/or, on the other hand, patientssuffering from auto-immune diseases for example (in the case in whichcatalytic antibodies are found in these patients).

Thus, according to a fourth principal aspect, the present inventionprovides a solution to a long-felt need through a pharmaceuticalcomposition characterised in that it comprises a pharmaceuticallyeffective amount of at least one anti-Factor VIII allo-antibody capableof degrading Factor VIII, as defined supra, notably as obtainable fromthe method described supra, or one of its pharmaceutically acceptableaddition salts incorporated in a pharmaceutically acceptable excipient,vehicle or carrier.

Further, according to a fifth principal aspect, the present inventionprovides a pharmaceutical composition characterised in that it comprisesa pharmaceutically effective amount of at least one Factor VIIIdegradation inhibitor, as defined supra, or one of its pharmaceuticallyacceptable addition salts incorporated in a pharmaceutically acceptableexcipient, vehicle or carrier.

These compositions can be administered by the buccal, rectal,parenteral, transdermal, ocular, nasal or auricular route, for example.

These compositions can be solid or liquid and can be presented in thepharmaceutical forms commonly used in human medicine, such as, forexample, simple or coated tablets, gelatine capsules, granules,suppositories, injectable preparations, transdermal systems, eye drops,aerosols and sprays, and ear drops. They are prepared by the customarymethods. The active principle, which consists of a pharmaceuticallyeffective amount of at least one Factor VIII degradation inhibitor asdefined supra, or one of its pharmaceutically acceptable addition saltscan be incorporated therein together with excipients normally employedin pharmaceutical compositions, such as talc, gum Arabic, lactose,starch, magnesium stearate, polyvidone, cellulose derivatives, cocoabutter, semi-synthetic glycerides, aqueous or non-aqueous vehicles, fatsof animal or vegetable origin, glycols, various wetting agents,dispersants or emulsifiers, silicone gels, certain polymers orcopolymers, preservatives, flavourings and colours. The preferredpharmaceutical form is an injectable form.

The invention also covers a pharmaceutical composition with neutralisingactivity which can be used especially as a favourable treatment ofdiseases such as haemophilia A with production of anti-Factor VIIIallo-antibodies; autoimmune diseases with anti-Factor VIIIallo-antibodies (in case catalytic antibodies are found in thesepatients) in particular, said composition being characterised in that itcomprises a pharmaceutically effective amount of at least one FactorVIII degradation inhibitor above, or one of its pharmaceuticallyacceptable addition salts incorporated in a pharmaceutically acceptableexcipient, vehicle or carrier.

The invention also covers a method of therapeutic treatment of a mammalsuffering from a pathology resulting from the level of Factor VIII inthe blood thereof, characterised in that a therapeutically effectiveamount of at least one Factor VIII degradation inhibitor as definedsupra or one of its pharmaceutically acceptable addition salts isadministered to the said mammal.

This method affords especially a favourable treatment of diseases ofhaemophilic nature, in particular a pathology resulting from a lack ofFactor VIII in the blood thereof.

The invention also covers a pharmaceutical composition withanti-thrombotic activity which can be used especially as a favourabletreatment of diseases such as thrombosis in particular, said compositionbeing characterised in that it comprises a pharmaceutically effectiveamount of at least one anti-Factor VIII allo-antibody capable ofdegrading Factor VIII, notably as obtainable from the method describedabove, or one of its pharmaceutically acceptable addition saltsincorporated in a pharmaceutically acceptable excipient, vehicle orcarrier.

The invention also covers a method of therapeutic treatment of mammals,characterised in that a therapeutically effective amount of at least oneanti-Factor VIII allo-antibody as defined supra or one of itspharmaceutically acceptable addition salts is administered to the saidmammal.

This method affords especially a favourable treatment of diseases ofthrombotic nature, in particular said pathology resulting from thepresence of an excess of Factor VIII in the blood thereof.

In human and animal therapeutics, the anti-Factor VIII allo-antibodiesor the Factor VIII degradation inhibitors as defined supra can beadministered by themselves or in association with a physiologicallyacceptable excipient, in any form, in particular orally in the form ofgelatine capsules or tablets, or parenterally in the form of injectablesolutions. It is possible to envisage other forms of administration suchas suppositories, ointments, creams, gels or aerosol preparations.

Within the context of the present invention, the following terms areused:

catalytic anti-Factor VIII allo-antibodies

, which is understood as meaning antibodies directed to Factor VIIIendowed with a catalytic activity induced in haemophilia A patients upontransfusion with therapeutic preparations of Factor VIII;

Factor VIII

, which is understood as meaning a co-enzyme of Factor IX in theenzymatic cleavage of Factor X during the blood coagulation process;

degradation of Factor VIII

, which is understood as meaning the generation of fragments from FactorVIII that do not appear due to a spontaneous hydrolysis, or due tohydrolysis by physiologically cleaving enzymes, i.e. thrombin, activatedFactor IX, activated Factor X, and activated protein C;

anti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor

, which is understood as meaning any peptide, belonging or not to theFactor VIII sequence, or protease inhibitor that are capable ofspecifically neutralising the hydrolysing activity of anti-Factor VIIIcatalytic antibodies;

DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION

Human recombinant Factor VIII was radio-labelled with ¹²⁵I. Anti-FactorVIII allo-antibodies were affinity-purified from the plasma of threehaemophilia patients with inhibitor on a Sepharose matrix to whichimmunopurified human Factor VIII had been coupled. Affinity-purifiedanti-Factor VIII antibodies of patients Bor, Che and Wal inhibitedFactor VIII pro-coagulant activity up to 57.0, 64.0 and 43.0 BU/mg ofIgG, respectively.

Co-incubation of labelled Factor VIII with the anti-Factor VIIIallo-antibodies resulted, in the case of two patients out of three, inthe proteolysis of the molecule. The specificity of the hydrolysis onthe antibody combining sites of anti-Factor VIII allo-antibodies of theIgG isotype was demonstrated. Co-incubation of [¹²⁵I]-Factor VIII withaffinity-purified anti-Factor VIII IgG of patients Bor and Wal in thepresence of the protease inhibitors aprotinin (0.15 μM), E-64 (28 μM),EDTA (1.3 μM), leupeptin (10 μM), and pepstatin (10 μM) did not resultin inhibition of proteolytic activity.

The Applicants have characterised the major cleavage sites for catalyticIgG in the Factor VIII molecule, to be as follows: Arg³⁷²-Ser³⁷³,located between the A1 and A2 domains of Factor VIII; Tyr¹⁶⁸⁰-Asp¹⁶⁸¹,located on the N-terminus of the A3 domain; and Glu¹⁷⁹⁴-Asp¹⁷⁹⁵ locatedwithin the A3 domain.

The time and dose-dependency of the hydrolysis of Factor VIII byanti-Factor VIII allo-antibodies has been demonstrated. In particular,hydrolysis was observed under conditions where anti-Factor VIII IgG andFactor VIII were co-incubated at molar ratios that were 80- to 9500-foldlower than those expected to be present in patients' plasma, suggestingthat hydrolysis is a mechanism of Factor VIII inactivation by thepatients' allo-antibodies in vivo.

The Applicants have further investigated the kinetics ofantibody-mediated hydrolysis of Factor VIII by incubating anti-FactorVIII IgG of patient Wal with increasing concentrations of unlabeledFactor VIII in the presence of a fixed concentration of [¹²⁵I]-FactorVIII. The curves of the reciprocal of the velocity plotted as a functionof the reciprocal of the substrate concentration were linear (r=0.99),suggesting that the reaction conformed to simple Michaelis-Mentenkinetics, as already observed for polyclonal catalytic auto-antibodies.The apparent catalytic efficiency, Vmax and rate of hydrolysis ofanti-Factor VIII allo-antibodies were calculated in the case of patientWal. The kinetic parameters of hydrolysis calculated in vitro, suggestthat proteolysis may be a mechanism of Factor VIII inactivation bypatients' allo-antibodies in vivo.

The association of Factor VIII with von Willebrand Factor (vWF)increases the catalytic rate of thrombin for Factor VIII, whereas itprotects Factor VIII from hydrolysis by activated protein C (APC). Theaddition of vWF to Factor VIII resulted in partial inhibition ofhydrolysis of Factor VIII by anti-Factor VIII IgG, i.e. 36.9%, whenpurified vWF and Factor VIII were mixed using a wt/wt ratio similar tothat present in normal plasma, i.e. 30 μg/ml of vWF versus 300 ng/ml ofFactor VIII.

The identification of anti-Factor VIII allo-antibodies as catalyticantibodies extends the spectrum of catalytic immune responses, inaddition to previous reports of hydrolysing antibodies againstvasoactive intestinal peptide (VIP) in asthma patients, DNA-hydrolysingantibodies in patients with SLĖ and thyroglobulin-specific catalyticantibodies in patients with autoimmune thyroiditis. This is also thefirst report to the knowledge of the Applicants of the induction of acatalytic antibody in the human, in response to exogeneousadministration of a protein antigen. The kinetic parameters of FactorVIII hydrolysis by anti-Factor VIII IgG exhibiting catalytic propertiesand the estimated amounts of these antibodies in plasma, suggest afunctional role for the catalytic immune response in inactivating FactorVIII in vivo. Within a polyclonal mixture of anti-Factor VIIIallo-antibodies which differ in their functional properties, catalyticantibodies may inhibit Factor VIII pro-coagulant activity at fasterrates than non-catalysing anti-Factor VIII antibodies. Identification ofpeptide epitopes that are the targets for proteolytic anti-Factor VIIIantibodies may thus be critical for our understanding of thepathophysiology of the Factor VIII inhibitor response. Furthermore, thecharacterisation of Factor VIII inhibitors as site-specific proteaseswill provide new approaches to the treatment of patients possessinganti-Factor VIII allo-antibodies.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be better understood and other objects,characteristics and advantages thereof will become more clearly apparentfrom the following explanatory description referring to the attachedFigures, which are given solely by way of non-limiting Examplesillustrating the specificity of the cleavage of Factor VIII byanti-Factor VIII allo-antibodies.

FIG. 1: Hydrolysis of [¹²⁵I]-Factor VIII by affinity-purifiedanti-Factor VIII IgG antibodies of haemophilia A patients with inhibitor

FIG. 1(A):

[125I]-labelled Factor VIII was incubated with affinity-purifiedanti-Factor VIII IgG of patients Bor (lane Bor), Che (lane Che) and Wal(lane Wal), or with buffer alone (lane 1) for 10 h at 38° C. prior toSDS-PAGE and autoradiography. In two of the three patients (Bor andWal), incubation of Factor VIII with affinity-purified anti-Factor VIIIIgG resulted in hydrolysis of the Factor VIII molecule. In contrast, themigration profile of Factor VIII was unchanged when [¹²⁵I]-labelledFactor VIII was incubated with anti-Factor VIII IgG purified from theplasma of patient Che (lane Che). The migration profile of Factor VIIIwas also unchanged upon incubation with human monoclonal M061anti-digoxin IgG (mAb) or with normal unfractionated polyclonal humanIgG (Sandoglobulin®, IVIg) that exhibit no inhibitory activity to FactorVIII.

FIG. 1(B):

Flow-throughs of the affinity columns were devoid of anti-Factor VIIIantibodies as determined by ELISA, and did not hydrolyse [¹²⁵I]-labelledFactor VIII.

FIG. 1(C):

Removal of IgG from the acid eluates containing affinity-purifiedanti-Factor VIII antibodies of patients Wal and Bor by chromatography onprotein G, resulted in the loss of their hydrolytic activity to FactorVIII.

FIG. 2: Size exclusion chromatography of the catalytic activity ofanti-Factor VIII antibodies

FIG. 2(A):

To further exclude the possibility that the proteolytic activity of theantibodies was due to contaminating proteases, affinity-purifiedanti-Factor VIII antibodies of patient Wal were treated with 8 M ureaand subjected to size exclusion chromatography. A major peak wasisolated in fraction 25 that corresponded to IgG as indicated by ELISA.The hydrolysing activity co-eluted with the IgG fraction and that theactivity was not detected in fractions in which IgG was not present(e.g., fraction 35).

FIG. 2(B):

The major peak that was isolated in fraction 25 corresponded to IgG asindicated by SDS-PAGE of the radio-labelled content of the fraction.

FIG. 3: Dose- and time-dependency of proteolysis of [¹²⁵I]-Factor VIIIby affinity-purified anti-Factor VIII antibodies of haemophilia Apatients with inhibitor.

The kinetics of the hydrolysis of Factor VIII by anti-Factor VIIIallo-antibodies of patients Bor and Wal. The rate of hydrolysis of[¹²⁵I]-labelled Factor VIII by anti-Factor VIII IgG of patient Wal wasfaster than that exhibited by anti-Factor VIII IgG of patient Bor,suggesting either that catalytic antibodies of the patients exhibitdifferent kinetic properties, or, alternatively, that the proportion ofcatalytic antibodies among the anti-Factor VIII antibodies differbetween the patients.

FIG. 4: Hydrolysis of [¹²⁵I]-Factor VIII by anti-Factor VIII IgGantibodies in the presence of increasing amounts of cold Factor VIII

Kinetics of antibody-mediated hydrolysis of Factor VIII by incubatinganti-Factor VIII IgG of patient Wal with increasing concentrations ofunlabelled Factor VIII in the presence of a fixed concentration of[¹²⁵I]-Factor VIII. The addition of increasing amounts of unlabelledFactor VIII resulted in dose-dependent inhibition of hydrolysis of[¹²⁵I]-Factor VIII by anti-Factor VIII IgG. Saturation of Factor VIIIhydrolysis was not attained with the maximum concentration of FactorVIII that was used (i.e. 1.7 μM). The curves of the reciprocal of thevelocity plotted as a function of the reciprocal of the substrateconcentration were linear (r=0.99), suggesting that the reactionconformed to simple Michaelis-Menten kinetics, as already observed forpolyclonal catalytic auto-antibodies.

FIG. 5: Inhibition of catalytic activity of anti-Factor VIII IgG ofpatient Wal

The proteolysis of radio-labelled Factor VIII by the anti-Factor VIIIallo-antibodies of patient Wal was inhibited to about 62% when theantibodies and Factor VIII were co-incubated in the presence ofPefabloc® (marketed by Roche Diagnostics GmbH, Mannheim, Germany),indicating the potency of certain serine protease inhibitor toneutralise the catalytic activity of some of the catalytic antibodies.

EXAMPLES Example I Affinity-Purification of Anti-Factor VIII Antibodies

Antibodies were isolated from plasma by ammonium sulphate precipitation.Antibodies reactive with Factor VIII were then affinity-purified on aCNBr-activated Sepharose 4B matrix to which immuno-purified commercialhuman plasma-derived Factor VIII had been coupled (25000 U/3 g of gel).The flow-throughs of the columns were collected. After extensive washingwith PBS pH 7.4, anti-Factor VIII antibodies were eluted using 0.2 Mglycine pH 2.8, dialysed against PBS and concentrated with Centriprep.Flow-throughs and eluates were aliquoted and stored at −20° C. untiluse. F(ab′)₂ fragments of anti-Factor VIII antibodies were prepared aspreviously described.

The concentration of anti-Factor VIII IgG was 130, 20 and 280 μg per 10mg of IgG applied to the column in the case of patients Bor, Che andWal, respectively, (i.e., 143±130 μg/mI of unfractionated plasma), whichis in agreement with previous observations.

Example II Factor VIII-Neutralising Activity

The Factor VIII-neutralising activity of anti-Factor VIII antibodies wasdetermined by the method of Kasper et al. and expressed as Bethesdaunits (BU) (ref). BU were defined as the inverse of the concentration ofIgG which causes 50% inhibition of Factor VIII procoagulant activity.Residual Factor VIII activity was measured in a one-stage assay bydetermination of the activated partial thromboplastin time using humanplasma depleted of Factor VIII (Behring) as substrate and humanplacental Pathromtin® (Behring) as activators. Heated plasma orimmunopurified anti-Factor VIII IgG to be tested, were incubated withpooled citrated human plasma for 2 h at 37° C. The clotting time of fourserial dilutions of a reference plasma pool (Immuno AG, Wien) wascompared with the clotting time of three dilutions of each sample to betested. Dilutions were carried out in Owren-Koller buffer (DiagnosticaStago). Inter-assay variation ranged between 1 and 2.5%.

Affinity-purified anti-Factor VIII antibodies of patients Bor, Che andWal inhibited Factor VIII pro-coagulant activity up to 57.0, 64.0 and43.0 BU/mg of IgG, respectively.

Example III Assay for Hydrolysis of Factor VIII

Commercial human recombinant Factor VIII was labelled with ¹²⁵I to aspecific activity of 11.6 nCi/μg, by using the iodogen method.[¹²⁵I]-Factor VIII (1.5 to 150 ng) was incubated in 50 μl of 50 mMtris-HCI pH 7.7, 100 mM glycine, 0.025% Tween-20 and 0.02% NaN₃ alone orwith 17 to 1667 nM of immuno-purified anti-Factor VIII IgG for 5 min to10 hours at 38° C. Human monoclonal anti-digoxin IgG M061 (mAb) andnormal unfractionated human polyclonal IgG (IVIg, Sandoglobulin®), wereused as negative controls. Samples were mixed 1:1 with Laemmli's bufferwithout mercaptoethanol, and were subjected to SDS electrophoresiswithout boiling, after loading 20 μl of each sample per lane. Sampleswere run in parallel on 7.5% and 15% SDS-PAGE under non-reducingconditions, after loading 20 μl of each sample per lane. Migration wasperformed at room temperature using a mini-PROTEAN II system at 25mA/gel, until the dye front reached the bottom of the gel. The gels werethen dried and protein bands revealed using X-OMAT AR. Followingautoradiography, the Factor VIII bands of apparent molecular weight 200and 300 kDa that are consistently hydrolysed by anti-Factor VIII IgG,were scanned so as to allow for the calculation of the rate ofhydrolysis of labelled Factor VIII.

Example IV Fast Protein Liquid Chromatography Gel Filtration

A hundred μl aliquot of anti-Factor VIII IgG of patient Wal (740 μg)treated with 8M urea was subjected to gel filtration on a superose-12column equilibrated with PBS-0.01% azide at a flow rate of 0.2 ml/min.Five hundred μl fractions were collected and assayed for the presence ofIgG by sandwich ELISA and for Factor VIII proteolytic activity afterten-fold dilution. The proteins in fraction 25 were radiolabelled with¹²⁵I and subjected to SDS-PAGE under non-reducing conditions in parallelwith normal polyclonal human IgG. The gel was stained with ComassieBlue, and also autoradiographed; both images were then overlaid. A majorpeak was isolated in fraction 25 that corresponded to IgG as indicatedby ELISA and SDS-PAGE of the radiolabelled content of the fraction. Thehydrolysing activity co-eluted with the IgG fraction and that theactivity was not detected in fractions in which IgG was not present(e.g., fraction 35).

Example V Analysis of NH₂-Terminal Sequences

Unlabelled human recombinant Factor VIII sucrose formulation (rDNA-BHK)(300 μg, octocog alfa, Bayer Corporation, Berkeley, Calif.) was treatedwith the anti-Factor VIII IgG of patient Wal (74 μg) in 1500 μl of 50 mMtris-HCI pH 7.7, 100 mM glycine, 0.025% tween-20 and 0.02% NaN₃ for 24hours at 38° C. The resultant Factor VIII fragments were run on a 10%SDS-PAGE at 50 mA under non-reducing conditions and transferred for 2hours at 100 mA on a Hybond-P PVDF membrane (Amersham, Little Chalfont,England) in 10 mM CAPS, 10% ethanol at pH 11.0. After staining withcoomassie blue, visible bands were cut and subjected to N-terminalsequencing, using an automatic protein microsequencer Prosize 492 cLC(PE-Applied Biosystems, Foster City, Calif.). The amount of proteinsequenced ranged from 0.5 to 2 pmoles, depending on the fragment.

The major scissile bonds were as follows: Arg³⁷²-Ser³⁷³ (R³⁷²-S³⁷³),located between the A1 and A2 domains of Factor VIII; Tyr¹⁶⁸⁰-Asp¹⁶⁸¹(Y¹⁶⁸⁰-D¹⁶⁸¹), located in the N-terminus of the A3 domain; andGlu¹⁷⁹⁴-Asp¹⁷⁹⁵ (E¹⁷⁹⁴-D¹⁷⁹⁵) located within the A3 domain. Multiplesite cleavage of Factor VIII by anti-Factor VIII antibodies mightoriginate from individual antibodies with polyspecific catalyticactivities or polyclonal populations of antibodies, each exhibiting aunique cleavage site specificity.

Amino acid sequence Cleavage site Ser Val Ala Lys Lys His Pro Arg³⁷² -Ser³⁷³ (SVAKKHP) (R³⁷²-S³⁷³) Asp Gln Arg Gln Gly Ala Glu Glu¹⁷⁹⁴-Asp¹⁷⁹⁵(DQRQGAE) (E¹⁷⁹⁴-D¹⁷⁹⁵) Asp Glu Asp Glu Asn Gln Sr Tyr¹⁶⁸⁰-Asp¹⁶⁸¹(DEDENQS) (Y¹⁶⁸⁰-D¹⁶⁸¹)

Example VI Inhibition Studies were Performed Using Pefabloc®, a GenericInhibitor of Serine Proteases

Hydrolysis of [¹²⁵I]-Factor VIII by affinity-purified anti-Factor VIIIIgG antibodies of haemophilia A patients with inhibitor in the presenceof Pefabloc®. [¹²⁵I]-Factor VIII (150 ng) was incubated alone, with 50μg/ml of immunopurified anti-Factor VIII IgG of patient Wal or in thepresence of both anti-Factor VIII IgG and 4 mM of the serine proteaseinhibitor Pefabloc® (Boehringer) for 5 h at 38° C. Factor VIII was thenanalysed by 7.5% SDS-PAGE under non-reducing conditions. Followingautoradiography, the Factor VIII bands of apparent molecular weight 200and 300 kDa that are consistently hydrolysed by anti-FVIII IgG, werescanned so as to allow for the calculation of the % of hydrolysis oflabelled Factor VIII.

The proteolysis of radiolabelled Factor VIII by the anti-Factor VIIIallo-antibodies of patient Wal was inhibited to about 62% when theantibodies and Factor VIII were co-incubated in the presence ofPefabloc®, indicating the potency of some serine protease inhibitor toneutralise the catalytic activity of some catalytic antibodies.

Further Observation

Upon screening the purified IgG of TEN high responder patients withhaemophilia A using ¹²⁵I-radiolabelled Factor VIII as the targetmolecule, a change was observed in the migration profile of Factor VIIIin the case of six patients. These results substantiate the Applicant'sprevious observations and indicate that catalytic anti-Factor VIIIantibodies are found in about 60% of the patients.

1-27. (canceled)
 28. An isolated amino acid sequence: SEQ. ID No. 1: SerVal Ala Lys Lys His Pro. 1   5


29. An isolated amino acid sequence: SEQ. ID No. 2: Asp Glu Asp Glu AsnGln Ser. 1   5


30. An isolated amino acid sequence: SEQ. ID No. 3: Asp Gln Arg Gln GlyAla Glu. 1   5


31. A peptide or non-peptide analogue of an amino acid sequence of claim28, which is capable of inhibiting any site in the Factor VIII moleculewhich is susceptible to being lysed by an anti-Factor VIIIallo-antibody.
 32. A peptide or non-peptide analogue of an amino acidsequence of claim 29, which is capable of inhibiting any site in theFactor VIII molecule which is susceptible to being lysed by ananti-Factor VIII allo-antibody.
 33. A peptide or non-peptide analogue ofan amino acid sequence of claim 30, which is capable of inhibiting anysite in the Factor VIII molecule which is susceptible to being lysed byan anti-Factor VIII allo-antibody.
 34. A method of neutralisingcatalytic anti-Factor VIII allo-antibodies comprising using ananti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor.
 35. The method of claim 34, wherein said inhibitor comprisesa protease inhibitor.
 36. The method of claim 35, wherein said proteaseinhibitor is 4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride.37. The method of claim 34, wherein said inhibitor inhibits cleavage ofthe scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1 and A2domains, Tyr¹⁶⁸⁰-Asp¹⁶⁸¹, located on the N-terminus of the A3 domain,and Glu¹⁷⁹⁴-Asp¹⁷⁹⁵ located within the A3 domain of the Factor VIIImolecule.
 38. The method of claim 34, wherein said inhibitor comprises apeptide or non-peptide analogue of the isolated amino acid sequence:SEQ. ID No. 1: Ser Val Ala Lys Lys His Pro. 1   5


39. The method of claim 34, wherein said inhibitor comprises a peptideor non-peptide analogue of the isolated amino acid sequence: SEQ. ID No.2: Asp Glu Asp Glu Asn Gln Ser. 1   5


40. The method of claim 34, wherein said inhibitor comprises a peptideor non-peptide analogue of the isolated amino acid sequence: SEQ. ID No.3: Asp Gln Arg Gln Gly Ala Glu. 1   5


41. A pharmaceutical composition which comprises a pharmaceuticallyeffective amount of a pharmaceutically active ingredient selected fromthe group consisting of an anti-Factor VIII allo-antibody capable ofdegrading Factor VIII, and a pharmaceutically acceptable salt thereof,in a pharmaceutically acceptable excipient, vehicle or carrier.
 42. Thepharmaceutical composition of claim 41, wherein said anti-Factor VIIIallo-antibody capable of degrading Factor VIII is as obtainable from amethod which comprises: i) isolating the plasma from a sample of bloodtaken from said mammal, ii) isolating anti-Factor VIII allo-antibodiesfrom said plasma; iii) placing said anti-Factor VIII allo-antibodies incontact with Factor VIII for a period of time sufficient to permit anydegradation of said Factor VIII by said anti-Factor VIIIallo-antibodies; and iv) determining, after said period of time, whethersaid Factor VIII has been degraded by said anti-Factor VIIIallo-antibodies.
 43. A method of therapeutic treatment of a mammalsuffering from a pathology resulting from abnormal level of Factor VIIIin the blood thereof, wherein a therapeutically effective amount of apharmaceutically active ingredient selected from the group consisting ofat least one anti-Factor VIII allo-antibody capable of degrading FactorVIII, and a pharmaceutically acceptable salt thereof, in apharmaceutically acceptable excipient, vehicle or carrier, isadministered to said mammal.
 44. The method of claim 43, wherein saidpathology results from the presence of an excess of Factor VIII in theblood thereof.
 45. The method of claim 44, wherein said pathology is ofthrombotic nature.
 46. The method of claim 45, which is a therapeutictreatment of a mammal suffering from thrombosis.
 47. A pharmaceuticalcomposition which comprises a pharmaceutically effective amount of apharmaceutically active ingredient selected from the group consisting ofa Factor VIII degradation inhibitor of claim 34, and a pharmaceuticallyacceptable salt thereof, in a pharmaceutically acceptable excipient,vehicle or carrier.
 48. A method of therapeutic treatment of a mammalsuffering from a pathology resulting from the sub-physiological level ofFactor VIII in the blood thereof, wherein a therapeutically effectiveamount of a pharmaceutically active ingredient selected from the groupconsisting of at least one Factor VIII degradation inhibitor, and apharmaceutically acceptable salt thereof, is administered to saidmammal.
 49. The method of claim 48, wherein said inhibitor comprises aprotease inhibitor.
 50. The method of claim 49, wherein said proteaseinhibitor is 4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride.51. The method of claim 48, wherein said inhibitor inhibits cleavage ofthe scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1 and A2domains, Tyr¹⁶⁸⁰-Asp¹⁶⁸¹, located on the N-terminus of the A3 domain,and Glu¹⁷⁹⁴-Asp¹⁷⁹⁵ located within the A3 domain of the Factor VIIImolecule.
 52. The method of claim 48, which comprises a peptide ornon-peptide analogue of the amino acid sequence: Ser Val Ala Lys Lys HisPro.


53. The method of claim 48, which comprises a peptide or non-peptideanalogue of the amino acid sequence: Asp Glu Asp Glu Asn Gln Ser.


54. The method of claim 48, which comprises a peptide or non-peptideanalogue of the amino acid sequence: Asp Gln Arg Gln Gly Ala Glu.


55. The method of claim 48, wherein said pathology is of haemophilicnature.
 56. The method of claim 55, wherein said pathology ofhaemophilic nature is a disease involving coagulation defects due toFactor VIII insufficiency.
 57. The method of claim 55, which is a methodof therapeutic treatment of a mammal suffering from haemophilia A. 58.An anti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor, which comprises a peptide or non-peptide analogue of theisolated amino acid sequence: SEQ. ID No. 1: Ser Val Ala Lys Lys HisPro. 1   5


59. An anti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor, which comprises a peptide or non-peptide analogue of theisolated amino acid sequence: SEQ. ID No. 2: Asp Glu Asp Glu Asn GlnSer. 1   5


60. An anti-Factor VIII allo-antibody-catalysed Factor VIII degradationinhibitor, which comprises a peptide or non-peptide analogue of theisolated amino acid sequence: SEQ. ID No. 3: Asp Gln Arg Gln Gly AlaGlu. 1   5


61. A pharmaceutical composition, which comprises a pharmaceuticallyeffective amount of an anti-Factor VIII allo-antibody-catalysed FactorVIII degradation inhibitor.
 62. The pharmaceutical composition of claim61, which comprises a protease inhibitor.
 63. The pharmaceuticalcomposition of claim 62, wherein said protease inhibitor is4-(2-aminoethyl)benzenesulphonyl fluoride hydrochloride.
 64. Thepharmaceutical composition of claim 61, wherein said inhibitor inhibitscleavage of the scissile bonds: Arg³⁷²-Ser³⁷³, located between the A1and A2 domains, Tyr¹⁶⁸⁰-Asp¹⁶⁸¹, located on the N-terminus of the A3domain, and Glu¹⁷⁹⁴-Asp¹⁷⁹⁵ located within the A3 domain of the FactorVIII molecule.
 65. The pharmaceutical composition of claim 61, whichcomprises a peptide or non-peptide analogue of the amino acid sequence:Ser Val Ala Lys Lys His Pro.


66. The pharmaceutical composition of claim 61, which comprises apeptide or non-peptide analogue of the amino acid sequence: Asp Glu AspGlu Asn Gln Ser.


67. The pharmaceutical composition of claim 61, which comprises apeptide or non-peptide analogue of the amino acid sequence: Asp Gln ArgGln Gly Ala Glu.


68. An isolated anti-Factor VIII allo-antibody, which has a catalyticactivity capable of catalysing degradation of Factor VIII.
 69. Anisolated anti-Factor VIII allo-antibody which is obtainable by a methodof determining the presence of anti-Factor VIII allow-antibodies capableof degrading Factor VIII in a mammal, which comprises: i) isolating theplasma from a sample of blood taken from said mammal, ii) isolatinganti-Factor VIII allo-antibodies from said plasma; v) placing saidanti-Factor VIII allo-antibodies in contact with Factor VIII for aperiod of time sufficient to permit any degradation of said Factor VIIIby said anti-Factor VIII allo-antibodies; and vi) determining, aftersaid period of time, whether said Factor VIII has been degraded by saidanti-Factor VIII allo-antibodies.
 70. The isolated anti-Factor VIIIallo-antibody of claim 68, which cleaves the following scissile bonds inthe Factor VIII molecule: Arg³⁷²-Ser³⁷³, located between the A1 and A2domains, Tyr¹⁶⁸⁰-Asp¹⁶⁸¹, located on the N-terminus of the A3 domain,and Glu¹⁷⁹⁴-Asp¹⁷⁹⁵ located within the A3 domain of the Factor VIIImolecule.
 71. The isolated anti-Factor VIII allo-antibody of claim 69,which cleaves the following scissile bonds in the Factor VIII molecule:Arg³⁷²-Ser³⁷³, located between the A1 and A2 domains, Tyr¹⁶⁸⁰-Asp¹⁶⁸¹,located on the N-terminus of the A3 domain, and Glu¹⁷⁹⁴-Asp¹⁷⁹⁵ locatedwithin the A3 domain of the Factor VIII molecule.